Method and kit for the quantitative and/or qualitative detection of components in a sample

ABSTRACT

A method and kit for quantitatively and/or qualitatively detecting one or more components in samples, including the use of metal-particle labelled reagents and an antibody conjugate are disclosed. The components are capable of binding to a probe. A kit and method for staining components in cell and tissue sections, based upon the detection method, are also disclosed.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/EP2003/009393, filed Aug. 25, 2003.

FIELD OF THE INVENTION

The present invention relates to the field of diagnostic assays and kitsfor the detection of biological molecules.

BACKGROUND TO THE INVENTION

Micro-arrays are tools for DNA and RNA molecular diagnostics. Detectionof nucleic acids is possible using parallelisation techniques therebyenabling the investigation of several thousands of sequences in onereaction or experiment. Most applications focus on expression profilingfor measuring adequately the expression of several thousands genes ofinterest. Detection of molecular binding on micro-arrays is visualizedby use of special fluorescent dyes such as Cy3 and Cy5. Visualization ofthe binding is limited by the stability of the fluorescent marker, andin order to evaluate the processed micro-arrays, highly sophisticatedand expensive laser scanning devices are required in addition to highlysophisticated software for analysing the data generated by laserscanning devices.

For the last thirty-five years, metal particles including gold andsilver have been used as both contrast enhancement agents or lightabsorption labels in many different types of analytic and/or diagnosticapplications. The great majority of these applications fall under thecategory of cytoimmunochemistry studies which have used gold or silverenhanced gold particles as markers to study structural aspects ofcellular, subcellular, or tissue organization. In these studies, metalparticles are usually detected and localized by electron microscopy,including scanning, transmission, and BEI (backscattered electronimaging). These methods take advantage of the electron dense nature ofmetals or the high atomic number of metals to facilitate the detectionof the gold particles by virtue of the large numbers of secondary andbackscattered electrons generated by the dense metal (see; Hayat,Immunogold-silver staining reference Page 1 and Chapters 1, 6-15; andHayat, Colloid Gold reference Chapters 1, 5, 7 and others).

A number of patents describe the use of enzymatic methods or gold-basedtechnology on micro-arrays to detect the presence of specific sequencesof DNA. PCT patent application number WO 00/72018 (EP 1 179 180)(Advanced Array Technologies) describes the use of biotinylated DNA asprobes against DNA samples immobilised on a glass micro-array, usinggold (10 nm) labelled streptavidin as a visualisation agent.

US 2001/0010906A1 describes optimisation of capture probes design forsandwich hybridisation on solid carrier.

EP 1 164 201 describes the use of inverted detection for identifyingand/or quantifying nucleotide target sequences on biochips usingmicro-fluidity techniques.

EP 1 096 024 describes a method for detection of homologue sequencesafter multiplex PCR for detecting Staphylococcus microorganisms.

AU8366001, AU7547501, CA2397280, WO0196604, AU736340, U.S. Pat. No.6,214,560, CN1282378T, EP1023456, EP1021554, AU1294399, WO9920789disclose a similar technique using gold labelled streptavidin particlesof at least 80 nm for visualisation of bound nucleic acids on a glassmicro-array using back scattered light.

U.S. Pat. No. 5,583,001, U.S. Pat. No. 196,306 and U.S. Pat. No.5,731,158 disclose the use of in situ amplification techniques whereinthe signal generated by the bound probe is amplified and visualisedusing enzymatic or gold based techniques. The catalysed amplificationreporter deposit (CARD) technique was found to result in a signalamplification ranging from 10 to 100 fold and found to give equalresults as the polymer based technique. The principle of CARD is widelyused for several applications including electron microscopy,immunohistochemistry, ELISA, and in situ hybridization. The use of goldin a CARD based amplification has been described for the above mentionedtechniques but real signal amplification on micro-arrays is notdisclosed using gold or an enzymatic based technology.

U.S. Pat. No. 6,451,980 discloses a technique for signal enhancement ofbi-specific antibody-polymer probe for use in immunoassay. Therein isdescribed the use of bispecific antibodies where one part of theantibody complex recognises the antigen and the other part binds thepolymer probe consisting of a poly-L-lysine backbone coated with“detectable signals”.

WO0206511 and AU8292001 (Genisphere) disclose an amplification techniqueapplicable on micro-arrays, consisting of a dendrimer-based approach.Dendrimers are DNA molecules marked with a fluorescent dye. The specialfeature is that those molecules can form three dimensional structures bya type of hybridisation which results in a supermolecule heavily markedwith Cy3 or Cy5 molecules. This leads to an enormous amplification ofthe signal generated by the bound nucleic acid of interest.

EP 1 230 396 and WO 01/36681 (Digene) disclose a technology detectingDNA/RNA hybrids on micro-arrays using a specific monoclonal antibodydirected specifically to RNA/DNA hybrids with visualisation usingfluorescent dyes.

WO 96/14314 disclose the use of a specific monoclonal antibody detectingDNA/PNA nucleic acid hybrids in solution and on a solid support.

For the detection of low concentrations of molecule components in thefield of diagnostics, the methods of chemiluminescence andelectrochemiluminescence are widely used. These methods provides a meansto detect low concentrations of components by amplifying the number ofluminescent molecules or photon generating events manyfold, theresulting “signal amplification” then allowing for detection of lowconcentration components. However, the above mentioned methods of signalamplification have associated limitations which makes the detection ofcomponents by these methods complicated, not easy to use, timeconsuming, and costly.

Problems of interference of chemical or enzymatic reactions,contamination, complicated and multi-step procedures, limitedadaptability to single step “homogeneous” (non-separation) formats, andthe requirement of costly and sophisticated instrumentation are areasthat those in the art are constantly trying to improve.

Improvements has so far failed to provide means for the quantativeand/or qualitative detection of molecules such as DNA, RNA, proteins,polypeptides without an evaluation step requiring an additional devicesuch as a laser scanner, equipment to measure scattered light and/orspecialised software. A low cost means of detection of components insamples is of importance in everyday fields of environmental science,veterinary medicine, pharmaceutical research, food and water qualitycontrol and the like. Furthermore a means which is simple to use,obviates the need for specialised training in equipment and/orprotocols. Furthermore, the detection of substances at lowconcentrations (less than about 1 picomole substance/sample volumeanalyzed) is presently not possible without the use of fluorescent,luminescent, chemiluminescent, or electrochemiluminescent labels andother detection methods, all of which require optical reading devices toevaluate results.

It is a purpose of this invention not only to overcome the present daylimitations, for example the disadvantages of light scattering-baseddiagnostic assays, but to also overcome the limitations anddisadvantages of other non-light scattering methods such as signalamplification. This invention as described herein is easier to use, hasgreater detection sensitivity, and is capable of measuring components insamples across a wider concentration range than was previously possible.The present invention is broadly applicable to most sample types andassay formats as a signal generation and detection system forcomponents.

The present invention provides a signal and detection system for thedetection of components where the procedures can be simplified and theamount and types of steps and reagents reduced. The present inventionprovides for the quantitative and/or qualitative detection of single ormultiple components in a sample. The present invention also provides forsubstantial reductions in the number of different tests and amounts ofsample material that are analysed. Such reduction in the number ofindividual tests leads to reduced cost and waste production, especiallymedically-related waste that must be disposed of.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for quantitativelyand/or qualitatively detecting one or more components in one or moresamples, said component capable of binding to a probe, comprising thesteps in the following order:

a) applying one or more samples onto a solid support,

b) optionally storing solid supports of step a) at a temperature between0 and 10 degrees Celsius,

c) incubating solid support of step a) or b) with one or more taggedprobes,

d) incubating solid support with a monoclonal or polyclonal antibodydirected against the tag of step c), said antibody raised in species Aand said antibody optionally labelled with metal particle,

e) incubating solid support with antibody conjugate, said polymercomprising:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,—one or more antibodies, anti-B, directed against        immunoglobulins of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative colour change compared with the solid        support,        f) incubating the solid support with a polypeptide capable        recognition by anti-B antibodies, said polypeptide labelled with        one or more substances which directly or indirectly cause a        quantitative colour change compared with the solid support, and        g) optionally incubating the solid support with a metal        enhancement reagent and/or a colour change reagent that is a        suitable substrate of an enzyme attached to the antibody        conjugate, and        h) reading the solid support to quantitatively and/or        qualitatively detect said components.

Another embodiment of the present invention is a method as describedabove wherein step a) is

-   -   a) applying one or more probes onto a solid support, and step c)        is    -   c) incubating solid supports with tag-labelled sample,

Another embodiment of the present invention is a method as describedabove wherein step c) is absent and step d) is

-   -   d) incubating solid supports with metal-particle-labelled        anti-component monoclonal or polyclonal antibody, said antibody        raised in species A.

Another embodiment of the present invention is a method as describedabove further comprising the steps, after step f), of:

-   -   f-1) repeating steps e) to f), and    -   f-2) optionally repeating step f-1).

Another embodiment of the present invention is a method as describedabove wherein the solid support is supplied with probe pre-applied, andstep a) is not performed by the user.

Another embodiment of the present invention is a method as describedabove wherein the reading of step h) comprises the use of a colourchart.

Another embodiment of the present invention is a method as describedabove wherein the reading of step h) comprises the use of a devicesuitable for detecting changes in conductance and/or current across thesolid support at the positions at which said samples are applied.

Another embodiment of the present invention is a kit for quantitativelyand/or qualitatively detecting one or more components in one or moresamples, said component capable of binding to a probe, comprising:

a) one or more solid supports,

b) a container in which a quantity antibody conjugate is present, saidconjugate comprising:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,—one or more antibodies, anti-B, directed against        immunoglobulins of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative colour change compared with the solid        support.

Another embodiment of the present invention is a kit as described abovefurther comprising a container in which a quantity of anti-tagpolyclonal or monoclonal antibodies is present, said antibodies raisedin species A.

Another embodiment of the present invention is a kit as described abovewherein the solid support is pre-loaded with probes capable of bindingto said components.

Another embodiment of the present invention is a kit as described abovefor use in a method as described above.

Another embodiment of the present invention is a kit according asdescribed above for use in detecting, diagnosing and/or monitoring theprogress of a Human Papillomavirus (HPV) infection and wherein one ormore molecular probes is capable of binding to an HPV component.

Another embodiment of the present invention is a kit as described abovewherein said component is a coat polypeptide.

Another embodiment of the present invention is a kit as described abovewherein said component is a gene selected from the group consisting ofHPV 16, HPV18, HPV 31, HPV 33, HPV 35, HPV 52 and HPV 58.

Another embodiment of the present invention is a kit as described abovefor use in detecting, diagnosing and/or monitoring the progress of oneor more of the disease states in humans as listed in Table 1, bydetecting a polypeptide and/or nucleic acid corresponding to the listedcomponent.

Another embodiment of the present invention is a kit as described abovefor use in detecting, diagnosing and/or monitoring the progressinfections caused by one or more of one or more of HCV, HIV, HBV, HTLV,mycobacteria, Staphylococcus aureus.

Another embodiment of the present invention is a kit as described abovefor use in detecting, diagnosing and/or monitoring the progressneurodegenerative diseases by detecting one or more of beta-amyloids,hTAU, phosphoTAU and APOE.

Another embodiment of the present invention is a kit as described abovefor use in detecting, diagnosing and/or monitoring the progress ofmalignant diseases, autoimmunity or allergy related diseases bydetecting one or more of ANA, Jo-1, Myeloperoxidase, RNP, Scl-70, Sm,SS-A, IgE, IgG-subclasses and circulating antibodies.

Another embodiment of the present invention is a kit as described abovefor use in environmental testing of water for bacteria.

Another embodiment of the present invention is a kit as described abovefor use in environmental testing of food components for geneticallymodified components, listeria and salmonella.

Another embodiment of the present invention is a method for stainingcomponents in cell and/or tissue sections suitable for visualisationusing microscopy comprising the steps of:

j) incubating said section with one or more tagged probes directedagainst a component,

k) incubating said section with metal labelled anti-tag monoclonal orpolyclonal antibody, said antibody raised in species A,

l) incubating said section with antibody/enzyme polymer, said polymercomprising at least:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,—one or more antibodies, anti-B, directed against        immunoglobulins of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative colour change,        m) incubating the section with a polypeptide capable recognition        by anti-B antibodies, said polypeptide labelled with one or more        substances which directly or indirectly cause a quantitative        colour change, and        n) optionally incubating the section with a metal enhancement        reagent and/or a colour change reagent that is a suitable        substrate of an enzyme attached to the antibody conjugate.

Another embodiment of the present invention is a method as describedabove wherein step j) is absent and step k) is

-   -   k) incubating section with metal particle labelled        anti-component monoclonal or polyclonal antibody, said antibody        raised in species A.

Another embodiment of the present invention is a method as describedabove further comprising the steps, after step m), of:

-   -   m-1) repeating steps l) to m), and    -   m-2) optionally repeating step m-1).

Another embodiment of the present invention is a kit for stainingcomponents in cell and/or tissue sections suitable for visualisationusing microscopy comprising:

a container in which a quantity of antibody/enzyme polymer antibody,said polymer comprising at least:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,—one or more antibodies, anti-B, directed against        immunoglobulins of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative colour change.

Another embodiment of the present invention is a kit as described abovefurther comprising a container in which a quantity of anti-tagpolyclonal or monoclonal antibodies is present, said antibodies raisedin species A.

Another embodiment of the present invention is a kit as described abovefor use in a method as described above.

Another embodiment of the present invention is a method as describedabove, and a kit according as described above wherein said metalparticle is gold.

Another embodiment of the present invention is a method as describedabove, and a kit as described above wherein said tag is biotin.

Another embodiment of the present invention is a method as describedabove, and a kit as described above wherein said polypeptide capablerecognition by anti-B antibodies is labelled with gold particles and/oralkaline phosphatase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to a method for the quantitative and/orqualitative detection of components in a sample, using a solid support,an antibody conjugate, a tagged probe directed to said components, andan antibody directed against said tag.

A “sample” as used herein means any sample that contains one or morecomponents to be tested, said components capable of binding to a probeenabling identification of said component. Examples of samples orcomponents include, but are not limited to DNA, cDNA, mRNA, RNA, nucleicacids, proteins, polypeptides, glycoproteins, receptors, ligands,metabolites, toxins etc. Other examples of samples include, but are notlimited to blood, blood components, other bodily fluids, tissues,drinking water, soil, domestic waste, industrial waste, any foodstuff—liquid or solid, crops.

A “probe” as used herein means any compound capable of specific bindingto a component. For example, a nucleic acid oligomer binding to a gene,a ligand binding to a receptor are examples of probe/componentinteractions according to the invention. According to the presentinvention, the affinity of binding between a probe and a component isbetter than 10 uM, 5 uM, 2 uM, 1 uM, 0.1 uM, 0.01 uM or 1 nM. Examplesof probes include but are not limited to nucleic acids, PNAs, proteins,peptides, antibodies, ligands, receptors etc.

A “tag” as used here means any type of substance which is capable ofbeing recognised by an antibody. Examples of such tags include, but arenot limited to polypeptides, proteins, polysaccharides, amino acids,vitamins (such as biotin), natural or synthetic substances, enzymes(such as AP and HRP), dyes (such as FITC and TR), nucleic acids, PNA,DNP, digoxygenin, streptavidin, Psoralen. A “tag” as used here alsomeans a metal, metals or organometallic substances. According to oneaspect of the invention, the probe is tagged.

According to another aspect of the invention, the sample is tagged. Theprocess of tagging is known to the skilled artisan and can be performedon proteins, peptides or nucleic acids. For example, nucleic acid may bebiotinylated by performing a polymerase chain reaction on the sampleusing biotinylated primer(s) specific for the gene of interest. Peptidesand proteins may be biotinylated using biotinylation reagents which, forexample, biotinylate the C-terminus, the N-terminus and/or reactive sidechains of the protein or peptide. The present invention includes anymethod of the art or future methods for the tagging of sample.

By “solid support” herein is meant any solid support which is capable ofimmobilising components and/or samples. Such solid supports are known inthe art and include, but are not limited to, nitrocellulose, glassslides, nylon, silane coated slides, nitrocellulose coated slides,plastics. The solid support preferably comprises nitrocellulose.

Antibodies as used according to the present invention may be wholeantibodies or may be part of an antibody, said part comprising at leastthe complementary determining region and, where necessary, aspecies-specific region that can be recognised by other antibodies (e.g.a constant region).

An “antibody conjugate” refers to a complex comprising a bridgingcompound with an antibody directed against antibodies raised in onespecies (anti-A, e.g. against part of the constant region of mouseantibodies) and/or an antibody directed against antibodies raised in adifferent species (anti-B, e.g. against part of the constant region ofrabbit antibodies). Attachment may be by covalent or non-covalent means.Examples of species against which said attached antibodies may be raisedinclude, but are not limited to rabbit, human, goat, mouse, rat, cow,calf, camel, llama, monkey, donkey, guinea pig, chicken and sheep.

According to an aspect of the invention the bridging compound is asoluble polymeric support such as, for example, dextran, polyethyleneglycol, agarose, acrylamide, protein, carbohydrate, any bio-polymer, andsynthetic polymer, nucleic acid, PNA, latex or any other known or futurepolymeric subustance. Optionally also attached to the bridging compoundare one or more substances that directly or indirectly cause aquantitative colour change compared with the solid support. Saidsubstances may be coloured, or are capable of inducing colour change.For example, the substance might be a dye, a metal particle, a non-metalparticle, or an enzyme that catalyses a colour-change reaction. It iswithin the scope of the invention that a combination of theaforementioned colour change substances are attached to the bridgingcompound.

A colour change according to the invention is a change in the colour orintensity of colour of the solid support at the location at which thesample is applied (e.g. from white to black, from white to red, fromwhite to grey, from white to blue, etc.)

The anti-tag antibody as used in the present invention can be anyantibody or part thereof containing a complementary-determining-regionthat is capable of binding to the tag. The anti-tag antibody mayoptionally be labelled with one or more substances that are coloured(e.g. a dye, a metal particle), or are capable of inducing colour change(e.g. an enzyme that catalyses a colour-change reaction.)

Examples of suitable dyes according to the invention, include but arenot limited to FITC, TR, Cy3, Cy5, Rhodamine, RPE, APC, DAPI, RPE-Cy5,PE, Fast Green, Alexins, Tamra, Joe, Rox, 6-FAM, HEX, TET, Dabcyl, TEG.

Examples of suitable metal particles according to the invention, includebut are not limited to gold, silver, iron, nickel, gadolinium, lead,uranium, caesium, platinum, rhodium, technetium, tellurium, selenium,silicon (silicium), cupper, tin, rhenium, europium, aluminium,germanium, chromium, cadmium, niobium, titanium, magnesium, manganese,molybdenum, antimony, americium, lithium, wolfram, and all metallicsubstances conducting or semi-conducting.

Enzymes that catalyse colour change and that are suitable according tothe invention may be any used in a colour change assay such as an ELISAor Western blot, and includes, but is not limited to alkalinephosphatase, horse radish peroxidase beta-galactosidase, luciferase,NADH. Reagents involved in visualising the colour change are known tothe skilled addressee and include, but are not limited to DAB, TMB,ABTS, AEC, OPD, Fast Red, fuchsine, AP-blue, AP-orange, BCIP, NBT, pNPP,BCI-NBT, CSPD, CDP-STAR, INT-BCIP.

The present invention is also related to the finding that the use ofmetal particle-labeled strepavidin or antibodies in a method of theinvention, said metal particle having a diameter 0.6 to 40 nm togetherwith metal enhancement, surprisingly leads to an improvement inquantitative and/or qualitative colour change, or conductance change.

The present invention is related to the finding that the use ofmetal-particle-labeled probes, said metal particle having a diameter 0.6to 40 nm and metal enhancement, in combination with the aforementionedconjugate for staining tissues and cells surprisingly leads to enhancedquantitative and/or qualitative colour change, or conductance change.

By metal enhancement is known to the skilled artisan in the field, andrefers to an enhancement of signal by means of metal precipitation dueto reduction. Means to enhance the signal include the use of metals suchas silver and nickel as secondary reagents. One embodiment of thepresent invention is a method for quantitatively and/or qualitativelydetecting one or more components in one or more samples, said componentcapable of binding to a probe, comprising the steps in the order of:

a) Applying one or more samples onto a solid support,

b) Optionally storing solid support at a temperature between 0 and 10degrees Celcius,

c) Incubating solid support with one or more tagged probes,

d) incubating solid support with a monoclonal or polyclonal antibodydirected against the tag of step c), said antibody raised in species A,and said antibody optionally labelled with metal particle,

e) Incubating spotted solid support with antibody conjugate, saidconjugate comprising:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,    -   one or more antibodies, anti-B, directed against immunoglobulins        of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative and/or qualitative colour change compared        with the solid support,        f) incubating the solid support of step e) with a polypeptide        capable of recognition by anti-B antibodies, said polypeptide        labeled with one or more substances which directly or indirectly        cause a quantitative colour change,        g) optionally incubating the solid support with a metal        enhancement reagent and/or a colour change reagent that is a        suitable substrate of an enzyme attached to the antibody        conjugate, and        h) reading the solid support to quantitatively and/or        qualitatively detect said components.

By “applying” as used herein in reference to applying one or moresamples or probes to a solid support, is meant deposition of one or moresynthetic or biological substances on a solid support. The depositionmay be by a manual method or by using a device, resulting in an actionincluding, but not limited to spotting, pipetting, printing, jetprinting, dropping etc.

By “reading” as used herein means determining from the change in theproperties of the solid support at the position where the sample orprobe is applied, the concentration of the components. According to theinvention a change in the property of the solid support may be a colourchange and/or a change in electrical conductivity or electrical currentat the position where the sample or probe is applied.

Reading may mean using normal vision to ascertain a colour change (e.g.from white to red, from white to black, from white to grey) on thematrix to determine the presence or absence of a component i.e. aqualitative reading. Reading may also mean using normal vision toascertain a colour change to determine the concentration of a componenti.e. a qualitative reading. It is within the scope of the invention thatthe reading may be taken using a colour chart that allows a comparisonof the colour of the sample with that of known concentrations of probeor component. It is within the scope of the invention that a colourchange disclosed herein may be read with or without the aid ofelectronic and optical measuring equipment. For example, a colour changeof the solid support may be read by means of a reflectance reader asdiscussed below.

Using a reflectance reader to measure a colour change leads to accuratemeasurements and allows the determination of the concentration of theprobe or component. The concentration of an unknown component can becalculated by interpolation on a standard curve obtained with severalconcentrations of probe or component.

Reading may also mean using a device to measure a change of electricalconductivity or electrical current at the position on the solid supportwhere the sample or probe is applied, to determine the concentration ofthe components. The inventors have found that the use of metal-labelledreagents according to the invention (e.g. metal-labeled antibodyconjugate, metal-labelled polypeptide capable of recognition by anti-Bantibodies) can result in a change of electrical conductivity orelectrical current of the solid support. The change can be convenientlyand accurately read using a device capable of detecting a change inconductivity and/or current across a solid support. Said device maycomprise one or more of the following features: one or more electricalcontact probes, circuitry to measure conductivity and/or current, ananalogue to digital converter. According to one example, one probe ofthe device contacts an upper surface of the solid support at theposition where the sample or probe is applied, and a second probecontacts the same position on the lower surface; the conductivity and/orcurrent across said probes is measured by the device. According toanother example, one probe of the device contacts an upper surface ofthe solid support at the position where the sample or probe is applied,and the whole of the lower surface of the solid support contacts aconducting plate; the conductivity and/or current across said probe andplate is measured by the device. The latter example has the conveniencethat the measurement of more than one sample requires movement of onlythe probe contacting the upper surface.

In one aspect of the invention, the samples are applied to the solidsupport without the addition of any extra reagents to the sample priorto the application of sample. In another aspect of the invention, thesamples are applied to the solid support after a preconditioningprocedure which increases the concentration of salt in said samples. Thesalt may be any dissociating salt in the art, including, but not limitedto sodium chloride, potassium chloride. The preconditioning may comprisethe addition of a volume of salt solution of a known concentration to avolume of sample. The preconditioning step may comprise the addition ofa volume of salt solution of a known concentration to an unknown volumeof sample. The concentration of salt in the sample may be adjusted tolie in the range of 100 mM to 500 mM, 500 mM to 1 M, 1 M to 1.5M, 1.5Mto 2M, 2M to 2.5M, 2.5M to 3M, 3M to 3.5M, 3.5M to 4 M, 3.5 M to 5 M,0.5 M to 2.5 M, 0.5 M to 3 M, 0.5 M to 4 M.

In another aspect of the invention, the sample is applied to the solidsupport at one or more positions with the same sample at the samerelative concentration. In another aspect of the invention, the sampleis applied to the solid support at one or more positions at thedifferent relative concentrations. In another aspect of the invention,the sample is applied to the solid support at one or more positions atthe same and/or different relative concentrations.

In another aspect of the invention, the temperature at which the solidsupport on which sample has been applied is optionally stored in step b)is between 0 and 10 degrees Celsius, 2 and 10 degrees, 3 and 10 degreesCelsius, 4 and 10 degrees Celsius, 5 and 10 degrees Celsius, 6 and 10degrees Celsius, 7 and 10 degrees Celsius, 0 and 5 degrees Celsius, 1and 5 degrees Celsius, 2 and 5 degrees Celsius, 3 and 5 degrees Celsius,1 degree Celsius, 2 degrees Celsius, 3 degrees Celsius, 4 degreesCelsius, 5 degrees Celsius, 6 degrees Celsius, 7 degrees Celsius, 8degrees Celsius, 9 degrees Celsius, 10 degrees Celsius.

In another aspect of the invention, when the metal particle is gold, thegold may be of any diameter. In another aspect, the gold may have anaverage diameter of 0.6, 0.8 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm,18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38nm, 39 nm, 40 nm and 0.6 to 1.0 nm, 1.0 to 5.0 nm, 5.0 to 10 nm, 10 to15 nm, 15 to 20 nm, 20 to 25 nm, 25 to 30 nm, 30 to 35 nm, 35 to 40 nm.

In another aspect of the invention, after applying the sample in stepa), step b) is performed without drying or baking the samples. Inanother aspect of the invention, the samples applied to the solidsupport in step a) are allowed to dry. Methods of drying samples areknown in the art and can include, but are not limited to, drying in theair, drying in a incubator, drying in an chamber under low pressureoptionally heated. According to another aspect of the invention, thesamples applied to the solid support are baked by exposed to atemperature of between 60 to 70 degrees Celsius, 65 to 75 degreesCelsius, 70 to 80 degrees Celsius, 75 to 85 degrees Celsius, 80 to 90degrees Celsius, 65 degrees Celsius, 70 degrees Celsius, 75 degreesCelsius, 80 degrees Celsius, 85 degrees Celsius or 90 degrees Celsius.The exposure time may be for no more than 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes or 60 minutes. The samples may be dried and thenbaked, only dried, only baked.

The introduction of wash steps in the method above, may be determined bythe skilled artisan in accordance with commonly understood protocols inimmunoassays such as ELISA and Western blots. For example, one or morewash steps may be introduced after one or more incubation step, usingwith a washing reagent such as a buffer. Examples of wash steps areprovided in the Examples section.

The use of an antibody conjugate as described herein leads to asurprising signal enhancement accompanied by an insignificant or absentincrease of background signal which could not have been anticipated bythe skilled artisan. Indeed, the inventors have tested other sandwichtype assays which do not use the antibody conjugate, and found anaccompanying increase in background signal and the introduction ofartefacts. The use of the antibody conjugate as disclosed herein, asfound by the inventors, provides signal amplification without aconcomitant increase in background signal.

The “metal enhancement reagent” of step g) is any metal-containingreagent wherein the metal precipitates due to reduction. Examplesinclude but are not limited to a silver enhancement reagent by Aurion(the Netherlands), BBI (UK), Sigma-Aldrich (USA), or Amersham (UK).

The “polypeptide capable of recognition by anti-B antibodies” in step f)can be any polypeptide or substance which is recognised by the anti-Bantibody attached to the antibody conjugate of step e). If it is apolypeptide, it might be an antibody raised in species B, or anantigenic part thereof. If it is another substance, it might bepolysaccharides, amino acid, natural or synthetic substances, nucleicacids, PNA, which is capable of binding to the complimentary determiningregion of the anti-B antibody of step e).

The components in the samples may be measured qualitatively orquantitatively determined by reading the solid support by measuring achange in property of the solid support at the position where the samplewas applied (e.g. measuring a colour change and/or a change inelectrical conductivity/current). The change in property may be causedby the dye, the metal, the metal after metal enhancement and/or thecolour produced after the enzyme catalyses a colour change reagent. Itis an aspect of the invention that the change in property is caused bythe metal without metal enhancement. It is an aspect of the inventionthat the change in property is caused by the dye alone. It is an aspectof the invention that the change in property is caused by metal usingmetal enhancement. It is an aspect of the invention that the change inproperty is caused by an enzyme-linked colour change. It is an aspect ofthe invention that the change in property is caused by one or more ofthe aforementioned aspects.

In another aspect of the invention, the use of tagged-probe as describedin a method above is circumvented by using an antibody raised in speciesA directly against a component in the sample. Thus, in this embodimentof the invention, step c) as described above is absent, and step d)reads:

d) incubating solid supports with metal-particle labelled anti-componentmonoclonal or polyclonal antibody, said antibody raised in species A.

The use of an antibody directed against a component in a sample obviatesthe need for an additional step in which a tagged probe is added, andfor an additional assay reagent, the introduction of which can lead tohandling error.

In another aspect of the invention, one or more probes are immobilizedonto the solid support and one or more samples applied thereto. Thus,the method is performed as described above including the variationsdisclosed, with steps substituted or performed identically, as indicatedin the method comprising the steps in the following order:

a) Applying one or more probes onto a solid support,

b) Optionally storing solid supports of step a) at 0 to 10 degreesCelsius,

c) Incubating solid support with tagged-sample,

d) Incubating solid support with metal-particle-labeled anti-tagmonoclonal or polyclonal antibody, said antibody raised in species A,

e) Incubating solid support with antibody conjugate, said conjugatecomprising:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,—one or more antibodies, anti-B, directed against        immunoglobulins of species B,    -   optionally one or more substances which directly or indirectly        cause a quantitative and/or qualitative colour change compared        with the solid support,        f) Incubating the solid support with a polypeptide capable        recognition by anti-B antibodies, said polypeptide labelled with        one or more substances which directly or indirectly cause a        quantitative colour change compared with the solid support, and        g) optionally incubating the solid support with a metal        enhancement reagent and/or a colour change reagent that is a        suitable substrate of an enzyme attached to the antibody        conjugate, and        h) reading the solid support to quantitatively and/or        qualitatively detect said components.

According to the above embodiment, the sample is tagged. The process oftagging is known to the skilled artisan and can be performed onproteins, peptides or nucleic acids as discussed above.

In another embodiment, the sample is not tagged, and the component boundto the probe is directly detected using a metal-labelled antibodydirected to said component. Thus, the need to tag sample is obviated. Inone embodiment of the invention, step c) as described above is absent,and step d) reads:

d) incubating solid supports with metal labelled anti-componentmonoclonal or polyclonal antibody, said antibody raised in species A.

In another embodiment of the present invention, the solid supports areprovided with the probe pre-applied. In one aspect of the invention, thesolid support is provided with probe located one or more positions, saidprobe recognising the same component. In one aspect of the invention,the solid support is provided with probe located one or more positions,said probe recognising different components. Thus, the method of theinvention wherein the probe is immobilised onto the solid support isperformed from step c). A method in which the solid support is providedwith probe pre-applied enables a sample to be assayed for componentswithout the necessity for performing probe application steps.Furthermore, a method using a solid support provided with probepre-applied, and probe recognising more than one component, enablessingle samples to be analysed for several components with a singleincubation. For example, a single solid support may be used to detectfor several cancerous or pre-cancerous conditions as described below byscreening a single sample.

It is one advantage of the invention that it does not necessarilyrequire an optical reading device such as a laser scanner orback-scatter measuring equipment, and hence is convenient for use inenvironments away from laboratory conditions. The invention allowsquantitative and/or qualitative results to be obtained at the locationat which the sample was taken, for example, in a general practitioner'ssurgery, in an individual's home, in hospitals, generally ‘in the field’without any specialist analytical instruments. Furthermore, theinvention provides an assay that is as sensitive as, or more sensitivethan assays which use fluorescence. Furthermore, since specialisedmeasuring equipment is not necessarily required, the assay could beperformed by a non-specialist.

Another embodiment of the present invention is a method for detecting acomponent in one or more samples, said method as describe above, furthercomprising, after step f) the steps of:

f-1) repeating steps e) to f), and

f-2) optionally repeating step f-1).

The steps described in the embodiment above are an amplification, whichfurther 30 enhances the sensitivity of the above method, whileminimizing the increase in background signal.

Another aspect of the present invention is a kit for the quantitativeand/or qualitative detection of components in a sample comprising thefollowing components:

k) one or more solid supports, and

l) a container in which a quantity of antibody conjugate as describedabove is present.

A container may be any sealed or resealable vessel suitable for carryinga quantity of antibody conjugate. Examples include, but are not limitedto screw cap vials, push cap vials, break-seal-to-open vials, syringes.

A kit according to the present invention allows a skilled artisan toperform one or more steps of the method disclosed herein, in aconvenient manner. The kit may allow a method of the present inventionto be performed without the need to measure or determine theconcentrations of reagents, so enabling a fast and reproducible assayingof one or more samples.

Another aspect of the present invention is a kit as described above,comprising a solid support according to item k), wherein said support ispre-spotted with one or more molecular probes. In one aspect of theinvention, the solid support is provided with probe located one or morepositions, said probe recognising the same component. In one aspect ofthe invention, the solid support is provided with probe located one ormore positions, said probe recognising different components. Thus, a kitsupplied with solid support in which the molecular probe is pre-appliedenables a sample to be assayed for components without the necessity forperforming application steps. Furthermore, a kit supplied with solidsupport pre-spotted with more than one molecule probe, each capable ofrecognising a different component enables a single sample to be analysedfor several components with a single incubation. For example, a singlesolid support may be used to detect for several pre-cancerous orcancerous conditions as described below by screening a single sample.

Another aspect of the present invention is a kit as disclosed herein,further comprising one or more metal-labelled probes. Each probe may bespecific for a component in a sample to be detected.

Another aspect of the present invention is a kit as disclosed herein,comprising antibody conjugate.

Another aspect of the present invention is a kit as disclosed herein,anti-tag antibody raised in species A, said antibody optionally labelledwith metal particle.

Another aspect of the present invention is a kit as disclosed herein, apolypeptide capable recognition by anti-B antibodies, said polypeptidelabelled with one or more substances which directly or indirectly causea quantitative colour change compared with the solid support.

Another aspect of the present invention is a kit as disclosed hereincomprising one or more additional containers in which reagent necessaryfor visualisation using the colour change enzyme linked to the antibodyconjugate are present.

Another aspect of the present invention is a kit as disclosed hereincomprising one or more additional containers in which reagent necessaryfor the tagging of sample to be tested are present. As already mentionedabove, method and reagent for tagging of protein and nucleic acids areknown in the art.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which metal-particle-labelledanti-component antibodies are present.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which a tagged probe(s) capable ofbinding to a component(s) is present.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which a polypeptide capablerecognition by anti-B antibodies, said polypeptide labelled with one ormore substances which directly or indirectly cause a quantitative colourchange compared with the solid support is present.

In another aspect of the invention, the kit enables the skilled personto perform one or more of the method disclosed herein. The kit maycomprise one or more additional containers in which reagents are presentenabling the skilled person to perform the complete method.

Alternatively, the kit may comprise a minimum number of containers, suchas only item 1), for example, that enables a skilled person to performthe method disclosed herein.

In another aspect of the invention, the kit contains instructions foruse. In another aspect of the invention the instructions describing amethod of the invention as disclosed herein.

In another aspect of the invention, the kit may be used for thediagnosis of disease, susceptibility of disease, monitoring the progressof disease, monitoring the progress of disease during treatment, testingof food, water, soil, testing for contamination, testing for thepresence of genetically modified (GM) food components and/or organisms.

Another aspect of the present invention is a method and/or kit asdisclosed herein for detecting the presence of a component in a sampleby visualisation, wherein the sample to be tested comprises a componentrelated to a disease, and the probe is an antibody directed against theDNA, mRNA, cDNA or polypeptide representing said component or partthereof in the diseased individual. Alternatively, the molecular probeis a nucleic acid (DNA, PNA) oligomer which is capable of hybridizing tothe DNA, mRNA, cDNA representing said component or part thereof in thediseased individual. A method and/or kit of the invention uses one ormore of the embodiments disclosed herein. Examples of components whichare associated with diseases and which are detectable using the methodand/or kit of the invention are provided in Table 1.

A method and/or kit according to the present invention may be used forthe diagnosis and detection of cancer in individuals, for example, forthe diagnosis of a type of cancer, for the early detection of cancer, tomonitor the progress of cancer in individuals already diagnosed with thedisease, to detect a relapse of cancer. Cancer is still a major diseaseand to prolong life expectancy, it would be advantageous to detect thedisease in a pre-clinical stage. A diagnostic assay as disclosed hereinmakes this possible. Non-limiting examples of components to which canceror several hereditary conditions associated with are provided in Table 1and, one or more of which are detectable using the method and/or kit ofthe invention. A diagnosis may require detection of one of more of thelisted molecules.

TABLE 1 List of components which are disease-related and are detectableusing the kit and/or method of the present invention. Number ComponentComments 1. BRCA1 breast cancer 1, early onset 2. TP53 tumor protein p53(Li-Fraumeni syndrome) 3. CFTR cystic fibrosis transmembrane conductanceregulator, ATP- binding cassette (sub-family C, member 7) 4. APP amyloidbeta (A4) precursor protein (protease nexin-II, Alzheimer disease) 5.APOE apolipoprotein E 6. BRCA2 breast cancer 2, early onset 7. HBBhemoglobin, beta 8. APC adenomatosis polyposis coli 9. MYC v-mycmyelocytomatosis viral oncogene homolog (avian) 10. HD huntington(Huntington disease) 11. BCL2 B-cell CLL/lymphoma 2 12. ABL1 v-ablAbelson murine leukemia viral oncogene homolog 1 13. BAX BCL2-associatedX protein 14. DMD dystrophin (muscular dystrophy, Duchenne and Beckertypes), includes DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269,DXS270, DXS272 15. CDKN2A cyclin-dependent kinase inhibitor 2A(melanoma, p16, inhibits CDK4) 16. ATM ataxia telangiectasia mutated(includes complementation groups A, C and D) 17. TNF tumor necrosisfactor (TNF superfamily, member 2) 18. RB1 retinoblastoma 1 (includingosteosarcoma) 19. VEGF vascular endothelial growth factor 20. ERBB2v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,neuro/glioblastoma derived oncogene homolog (avian) 21. FGG fibrinogen,gamma polypeptide 22. HPRT1 hypoxanthine phosphoribosyltransferase 1(Lesch-Nyhan syndrome) 23. MAPT microtubule-associated protein tau 24.MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein(mouse) 25. RUNX1 runt-related transcription factor 1 (acute myeloidleukemia 1; aml1 oncogene) 26. SOD1 superoxide dismutase 1, soluble(amyotrophic lateral sclerosis 1 (adult)) 27. CDKN1A cyclin-dependentkinase inhibitor 1A (p21, Cip1) 28. PAX6 paired box gene 6 (aniridia,keratitis) 29. NF1 neurofibromin 1 (neurofibromatosis, vonRecklinghausen disease, Watson disease) 30. FN1 fibronectin 1 31. CASP3caspase 3, apoptosis-related cysteine protease 32. PAH phenylalaninehydroxylase 33. GAPD glyceraldehyde-3-phosphate dehydrogenase 34. PTENphosphatase and tensin homolog (mutated in multiple advanced cancers 1)35. HFE hemochromatosis 36. FGFR3 fibroblast growth factor receptor 3(achondroplasia, thanatophoric dwarfism) 37. EGFR epidermal growthfactor receptor (erythroblastic leukemia viral (v-erb-b) oncogenehomolog, avian) 38. DSCR1 Down syndrome critical region gene 1 39. MLH1mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) 40. PABPC1poly(A) binding protein, cytoplasmic 1 41. CYP3A5 cytochrome P450,subfamily IIIA (niphedipine oxidase), polypeptide 5 42. PSEN1 presenilin1 (Alzheimer disease 3) 43. FBN1 fibrillin 1 (Marfan syndrome) 44. MSH2mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) 45. AKT1v-akt murine thymoma viral oncogene homolog 1 46. CCND1 cyclin D1(PRAD1: parathyroid adenomatosis 1) 47. MTHFR5,10-methylenetetrahydrofolate reductase (NADPH) 48. AR androgenreceptor (dihydrotestosterone receptor; testicular feminization; spinaland bulbar muscular atrophy; Kennedy disease) 49. TGFB1 transforminggrowth factor, beta 1 (Camurati-Engelmann disease) 50. IL6 interleukin 6(interferon, beta 2) 51. KRAS2 v-Ki-ras2 Kirsten rat sarcoma 2 viraloncogene homolog 52. HRAS v-Ha-ras Harvey rat sarcoma viral oncogenehomolog 53. RET ret proto-oncogene (multiple endocrine neoplasia andmedullary thyroid carcinoma 1, Hirschsprung disease) 54. PPARGperoxisome proliferative activated receptor, gamma 55. ACTB actin, beta56. CDH1 cadherin 1, type 1, E-cadherin (epithelial) 57. ESR1 estrogenreceptor 1 58. IGF1 insulin-like growth factor 1 (somatomedin C) 59.GSTP1 glutathione S-transferase pi 60. IL8 interleukin 8 61. LPLlipoprotein lipase 62. FMR1 fragile X mental retardation 1 63. WT1 Wilmstumor 1 64. IL1B interleukin 1, beta 65. CYP1A1 cytochrome P450,subfamily I (aromatic compound- inducible), polypeptide 1 66. CTNNB1catenin (cadherin-associated protein), beta 1 (88 kD) 67. ITGA5integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 68. FOSv-fos FBJ murine osteosarcoma viral oncogene homolog 69. KIT v-kitHardy-Zuckerman 4 feline sarcoma viral oncogene homolog 70. ATP7BATPase, Cu++ transporting, beta polypeptide (Wilson disease) 71. IGF2insulin-like growth factor 2 (somatomedin A) 72. JUN v-jun sarcoma virus17 oncogene homolog (avain) 73. CYP2C19 cytochrome P450, subfamily IIC(mephenytoin 4- hydroxylase), polypeptide 19 74. BCR breakpoint clusterregion 75. FGFR2 fibroblast growth factor receptor 2 (bacteria-expressedkinase, keratinocyte growth factor receptor, craniofacial dysostosis 1,Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) 76. CASP8caspase 8, apoptosis-related cysteine protease 77. INSR insulin receptor78. G6PD glucose-6-phosphate dehydrogenase 79. IL4 interleukin 4 80.DRD2 dopamine receptor D2 81. FGFR1 fibroblast growth factor receptor 1(fms-related tyrosine kinase 2, Pfeiffer syndrome) 82. COL1A1 collagen,type I, alpha 1 83. BLM Bloom syndrome 84. NF2 neurofibromin 2(bilateral acoustic neuroma) 85. MMP1 matrix metalloproteinase 1(interstitial collagenase) 86. IL2 interleukin 2 87. GRB2 growth factorreceptor-bound protein 2 88. BCL2L1 BCL2-like 1 89. PSEN2 presenilin 2(Alzheimer disease 4) 90. TNFRSF6 tumor necrosis factor receptorsuperfamily, member 6 91. CD44 CD44 antigen (homing function and Indianblood group system) 92. MMP9 matrix metalloproteinase 9 (gelatinase B,92 kD gelatinase, 92 kD type IV collagenase) 93. ABCB1 ATP-bindingcassette, sub-family B (MDR/TAP), member 1 94. GSTM1 glutathioneS-transferase M1 95. IL1A interleukin 1, alpha 96. MET metproto-oncogene (hepatocyte growth factor receptor) 97. ABO ABO bloodgroup (transferase A, alpha 1-3-N- acetylgalactosaminyltransferase;transferase B, alpha 1-3- galactosyltransferase) 98. NRAS neuroblastomaRAS viral (v-ras) oncogene homolog 99. NAT2 N-acetyltransferase 2(arylamine N-acetyltransferase) 100. EGR1 early growth response 1 101.TTR transthyretin (prealbumin, amyloidosis type I) 102. SOD2 superoxidedismutase 2, mitochondrial 103. SCYA2 small inducible cytokine A2(monocyte chemotactic protein 1) 104. NOS3 nitric oxide synthase 3(endothelial cell) 105. CDC2 cell division cycle 2, G1 to S and G2 to M106. STAT1 signal transducer and activator of transcription 1, 91 kD107. SNCA synuclein, alpha (non A4 component of amyloid precursor) 108.CLU clusterin (complement lysis inhibitor, SP-40, 40, sulfatedglycoprotein 2, testosterone-repressed prostate message 2,apolipoprotein J) 109. CDKN1B cyclin-dependent kinase inhibitor 1B (p27,Kip1) 110. TYR tyrosinase (oculocutaneous albinism IA) 111. MADH4 MAD,mothers against decapentaplegic homolog 4 (Drosophila) 112. CDK2cyclin-dependent kinase 2 113. MMP3 matrix metalloproteinase 3(stromelysin 1, progelatinase) 114. YWHAZ tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide 115. CASP1 caspase 1, apoptosis-related cysteine protease(interleukin 1, beta, convertase) 116. PCNA proliferating cell nuclearantigen 117. HLA-A, -B, -C major histocompatibility complex, class I, A,B, C 118. APOB apolipoprotein B (including Ag(x) antigen) 119. CASP9caspase 9, apoptosis-related cysteine protease 120. NOS2A nitric oxidesynthase 2A (inducible, hepatocytes) 121. IFNG interferon, gamma 122.APOA1 apolipoprotein A–I 123. AGT angiotensinogen (serine (or cysteine)proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 8) 124. ADA adenosine deaminase 125. ICAM1 intercellular adhesionmolecule 1 (CD54), human rhinovirus receptor 126. CYP19 cytochrome P450,subfamily XIX (aromatization of androgens) 127. SLC6A4 solute carrierfamily 6 (neurotransmitter transporter, serotonin), member 4 128.TNFRSF1A tumor necrosis factor receptor superfamily, member 1A 129. CD4CD4 antigen (p55) 130. VWF von Willebrand factor 131. ACTA1 actin, alpha1, skeletal muscle 132. MECP2 methyl CpG binding protein 2 (Rettsyndrome) 133. COMT catechol-O-methyltransferase 134. TERT telomerasereverse transcriptase 135. PKD polycystic kidney disease 1 (autosomaldominant) 136. F7 coagulation factor VII (serum prothrombin conversionaccelerator) 137. PMP22 peripheral myelin protein 22 138. F5 coagulationfactor V (proaccelerin, labile factor) 139. PPARA peroxisomeproliferative activated receptor, alpha 140. GCK glucokinase (hexokinase4, maturity onset diabetes of the young 2) 141. MUC1 mucin 1,transmembrane 142. SPP1 secreted phosphoprotein 1 (osteopontin, bonesialoprotein I, early T-lymphocyte activation 1) 143. RAF1 v-raf-1murine leukemia viral oncogene homolog 1 144. IGF1R insulin-like growthfactor 1 receptor 145. IL4R interleukin 4 receptor 146. DCC deleted incolorectal carcinoma 147. PML promyelocytic leukemia 148. PDGFRBplatelet-derived growth factor receptor, beta polypeptide 149. AGTR1angiotensin receptor 1 150. UBE3A ubiquitin protein ligase E3A (humanpapilloma virus E6- associated protein, Angelman syndrome) 151. CREBBPCREB binding protein (Rubinstein-Taybi syndrome) 152. CYP1B1 cytochromeP450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma 3, primaryinfantile) 153. AKT2 v-akt murine thymoma viral oncogene homolog 2 154.PLAT plasminogen activator, tissue 155. CHRNA7 cholinergic receptor,nicotinic, alpha polypeptide 7 156. TIMP1 tissue inhibitor ofmetalloproteinase 1 (erythroid potentiating activity, collagenaseinhibitor) 157. NFKB1 nuclear factor of kappa light polypeptide geneenhancer in B-cells 1 (p105) 158. STAT3 signal transducer and activatorof transcription 3 (acute- phase response factor) 159. CDC42 celldivision cycle 42 (GTP binding protein, 25 kD) 160. VDR vitamin D(1,25-dihydroxyvitamin D3) receptor 161. NTRK1 neurotrophic tyrosinekinase, receptor, type 1 162. VIM vimentin 163. TGFBR2 transforminggrowth factor, beta receptar II (70–80 kD) 164. DHFR dihydrofolatereductase 165. PTCH patched homolog (Drosophila) 166. CYP2A6 cytochnomeP450, subfamily IIA (phenobarbital-inducible), polypeptide 6 167. HSPCAheat shock 90 kD protein 1, alpha 168. E2F1 E2F transcription factor 1169. CACNA1A calcium channel, voltage-dependent, P/Q type, alpha 1Asubunit 170. LCK lymphocyte-specific protein tyrosine kinase 171. LGALS3lectin, galactoside-binding, soluble, 3 (galectin 3) 172. RARA retinoicacid receptor, alpha 173. PDZK1 PDZ domain containing 1 174. ALDH2aldehyde dehydrogenase 2 family (mitochondrial) 175. PAX3 paired boxgene 3 (Waardenburg syndrome 1) 176. FGF2 fibroblast growth factor 2(basic) 177. GJB1 gap junction protein, beta 1, 32 kD (connexin 32,Charcot- Marie-Tooth neuropathy, X-linked) 178. LMNA lamin A/C 179.CAPN3 calpain 3, (p94) 180. ADPRT ADP-ribosyltransferase (NAD+; poly(ADP-ribose) polymerase) 181. TUBB tubulin, beta polypeptide 182. ABCA1ATP-binding cassette, sub-family A (ABC1), member 1 183. IL1RNinterleukin 1 receptor antagonist 184. CTGF connective tissue growthfactor 185. GSTT1 glutathione S-transferase theta 1 186. DRD4 dopaminereceptor D4 187. HTR2A 5-hydroxytryptamine (serotonin) receptor 2A 188.FHIT fragile histidine triad gene 189. ETV6 ets variant gene 6 (TELoncogene) 190. PDGFB platelet-derived growth factor beta polypeptide(simian sarcoma viral (v-sis) oncogene homolog) 191. PPP3R1 proteinphosphatase 3 (formerly 2B), regulatory subunit B (19 kD), alpha isoform(calcineurin B, type I) 192. TIMP3 tissue inhibitor of metalloproteinase3 (Sorsby fundus dystrophy, pseudoinflammatory) 193. COL1A2 collagen,type I, alpha 2 194. ITGB3 integrin, beta 3 (platelet glycoprotein IIIa,antigen CD61) 195. COL3A1 collagen, type III, alpha 1 (Ehlers-Danlossyndrome type IV, autosomal dominant) 196. ESR2 estrogen receptor 2 (ERbeta) 197. B2M beta-2-microglobulin 198. SDF1 stromal cell-derivedfactor 1 199. F9 coagulation factor IX (plasma thromboplastic component,Christmas disease, hemophilia B) 200. MAPK14 mitogen-activated proteinkinase 14 201. BAK1 BCL2-antagonist/killer 1 202. ITGB1 integrin, beta 1(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12) 203. ACTG1 actin, gamma 1 204. KDR kinase insert domain receptor(a type III receptor tyrosine kinase) 205. SCTR secretin receptor 206.LEPR leptin receptor 207. SP1 Sp1 transcription factor 208. CDKN1Ccyclin-dependent kinase inhibitor 1C (p57, Kip2) 209. MYCN v-mycmyelocytomatosis viral related oncogene, neuroblastoma derived (avian)210. IiIL12B interleukin 12B (natural killer cell stimulatory factor 2,cytotoxic lymphocyte maturation factor 2, p40) 211. IGF2R insulin-likegrowth factor 2 receptor 212. FLT1 fms-related tyrosine kinase 1(vascular endothelial growth factor/vascular permeability factorreceptor) 213. CD36 CD36 antigen (collagen type I receptor,thrombospondin receptor) 214. FRD Friedreich ataxia 215. COL2A1collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphysealdysplasia, congenital) 216. GSN gelsolin (amyloidosis, Finnish type)217. CYP2E cytochrome P450, subfamily IIE (ethanol-inducible) 218. APAF1apoptotic protease activating factor 219. ANK1 ankyrin 1, erythrocytic220. SLC6A3 solute carrier family 6 (neurotransmitter transporter,dopamine), member 3 221. CASP7 caspase 7, apoptosis-related cysteineprotease 222. MYH7 myosin, heavy polypeptide 7, cardiac muscle, beta223. JUNB jun B proto-oncogene 224. GHR growth hormone receptor 225.IRS1 insulin receptor substrate 1 226. CASP10 caspase 10,apoptosis-related cysteine protease 227. BDNF brain-derived neurotrophicfactor 228. ATP7A ATPase, Cu++ transporting, alpha polypeptide (Menkessyndrome) 229. TCF1 transcription factor 1, hepatic; LF-B1, hepaticnuclear factor (HNF1), albumin proximal factor 230. HGF hepatocytegrowth factor (hepapoietin A; scatter factor) 231. CYP17 cytochromeP450, subfamily XVII (steroid 17-alpha- hydroxylase), adrenalhyperplasia 232. PTPN1 protein tyrosine phosphatase, non-receptor type 1233. ADRB3 adrenergic, beta-3-, receptor 234. TNFSF6 tumor necrosisfactor (ligand) superfamily, member 6 235. ERCC5 excision repaircross-complementing rodent repair deficiency, complementation group 5(xeroderma pigmentosum, complementation group G (Cockayne syndrome))236. VCAM1 vascular cell adhesion molecule 1 237. TF transferrin 238.ACE angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 239. LRP1low density lipoprotein-related protein 1 (alpha-2- macroglobulinreceptor) 240. CDK5 cyclin-dependent kinase 5 241. ACACA acetyl-CoenzymeA carboxylase alpha 242. TNFRSF1B tumor necrosis factor receptorsuperfamily, member 1B 243. NOTCH3 Notch homolog 3 (Drosophila) 244.ERBB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)245. CSK c-src tyrosine kinase 246. SCN5A sodium channel, voltage-gated,type V, alpha polypeptide (long (electrocardiographic) QT syndrome 3)247. BCL6 B-cell CLL/lymphoma 6 (zinc finger protein 51) 248. FYN FYNoncogene related to SRC, FGR, YES 249. CTSK cathepsin K(pycnodysostosis) 250. SPARC secreted protein, acidic, cysteine-rich(osteonectin) 251. NFKB2 nuclear factor of kappa light polypeptide geneenhancer in B-cells 2 (p49/p100) 252. SCYA5 small inducible cytokine A5(RANTES) 253. BMP4 bone morphogenetic protein 4 254. ATP2A2 ATPase, Ca++transporting, cardiac muscle, slow twitch 2 255. NR3C1 nuclear receptorsubfamily 3, group C, member 1 256. THBS1 thrombospondin 1 257. CETPcholesteryl ester transfer protein, plasma 258. PTPRC protein tyrosinephosphatase, receptor type, C 259. NME1 non-metastatic cells 1, protein(NM23A) expressed in 260. TGFBI transforming growth factor,beta-induced, 68 kD 261. SREBF1 sterol regulatory element bindingtranscription factor 1 262. MMP14 matrix metalloproteinase 14(membrane-inserted) 263. KCNQ1 potassium voltage-gated channel, KQT-likesubfamily, member 1 264. TUBA1 tubulin, alpha 1 (testis specific) 265.SELE selectin E (endothelial adhesion molecule 1) 266. ATRX alphathalassemia/mental retardation syndrome X-linked (RAD54 homolog, S.cerevisiae) 267. IL2RG interleukin 2 receptor, gamma (severe combinedimmunodeficiency) 268. IGFBP3 insulin-like growth factor binding protein3 269. JAK3 Janus kinase 3 (a protein tyrosine kinase, leukocyte) 270.CSF1R colony stimulating factor 1 receptor, formerly McDonough felinesarcoma viral (v-fms) oncogene homolog 271. SHC1 SHC (Src homology 2domain containing) transforming protein 1 272. CASP4 caspase 4,apoptosis-related cysteine protease 273. PLA2G2A phospholipase A2, groupIIA (platelets, synovial fluid) 274. CXCR4 chemokine (C—X—C motif),receptor 4 (fusin) 275. CDKN2B cyclin-dependent kinase inhibitor 2B(p15, inhibits CDK4) 276. ARHA ras homolog gene family, member A 277.SHH sonic hedgehog homolog (Drosophila) 278. RARB retinoic acidreceptor, beta 279. MME membrane metallo-endopeptidase (neutralendopeptidase, enkephalinase, CALLA, CD10) 280. CA2 carbonic anhydraseII 281. PRKDC protein kinase, DNA-activated, catalytic polypeptide 282.HIF1A hypoxia-inducible factor 1, alpha subunit (basic helix-loop- helixtranscription factor) 283. PRKCA protein kinase C, alpha 284. CASP2caspase 2, apoptosis-related cysteine protease (neural precursor cellexpressed, developmentally down-regulated 2) 285. DMBT1 deleted inmalignant brain tumors 1 286. TGFB2 transforming growth factor, beta 2287. TSC2 tuberous sclerosis 2 288. PSAP prosaposin (variant Gaucherdisease and variant metachromatic leukodystrophy) 289. XPC xerodermapigmentosum, complementation group C 290. THRA thyroid hormone receptor,alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian)291. ERCC2 excision repair cross-complementing rodent repair deficiency,complementation group 2 (xeroderma pigmentosum D) 292. MAPK1mitogen-activated protein kinase 1 293. ATP6B1 ATPase, H+ transporting,lysosomal (vacuolar proton pump), beta polypeptide, 56/58 kD, isoform 1(Renal tubular acidosis with deafness) 294. BAG1 BCL2-associatedathanogene 295. ACHE acetylcholinesterase (YT blood group) 296. EGFepidermal growth factor (beta-urogastrone) 297. DUSP1 dual specificityphosphatase 1 298. CASP6 caspase 6, apoptosis-related cysteine protease299. THRB thyroid hormone receptor, beta (erythroblastic leukemia viral(v-erb-a) oncogene homolog 2, avian) 300. BAD BCL2-antagonist of celldeath 301. STAT6 signal transducer and activator of transcription 6,interleukin-4 induced 302. ELN elastin (supravalvular aortic stenosis,Williams-Beuren syndrome) 303. MAOA monoamine oxidase A 304. F8coagulation factor VIII, procoagulant component (hemophilia A) 305. ENGendoglin (Osler-Rendu-Weber syndrome 1) 306. HSPB1 heat shock 27 kDprotein 1 307. HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase308. PIM1 pim-1 oncogene 309. PON1 paraoxonase 1 310. AHR arylhydrocarbon receptor 311. ITGB2 integrin, beta 2 (antigen CD18 (p95),lymphocyte function- associated antigen 1; macrophage antigen 1 (mac-1)beta subunit) 312. PTGS1 prostaglandin-endoperoxide synthase 1(prostaglandin G/H synthase and cyclooxygenase) 313. PLCG1 phospholipaseC, gamma 1 (formerly subtype 148) 314. APOC3 apolipoprotein C-III 315.NRG1 neuregulin 1 316. CD14 CD14 antigen 317. IRF1 interferon regulatoryfactor 1 318. ALPL alkaline phosphatase, liver/bone/kidney 319. ALDOAaldolase A, fructose-bisphosphate 320. XPA xeroderma pigmentosum,complementation group A 321. PDGFRA platelet-derived growth factorreceptor, alpha polypeptide 322. IL5 interleukin 5 (colony-stimulatingfactor, eosinophil) 323. BMP2 bone morphogenetic protein 2 324. GSK3Aglycogen synthase kinase 3 alpha 325. STK11 serine/threonine kinase 11(Peutz-Jeghers syndrome) 326. GSK3B glycogen synthase kinase 3 beta 327.CRYBB1 crystallin, beta B1 328. STAT5A signal transducer and activatorof transcription 5A 329. SCA1 spinocerebellar ataxia 1(olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1) 330. RXRAretinoid X receptor, alpha 331. NFKBIA nuclear factor of kappa lightpolypeptide gene enhancer in B-cells inhibitor, alpha 332. MMP13 matrixmetalloproteinase 13 (callagenase 3) 333. TSHR thyroid stimulatinghormone receptor 334. MT2A metallothionein 2A 335. TSSC3 tumorsuppressing subtransferable candidate 3 336. RHO rhodopsin (opsin 2, rodpigment) (retinitis pigmentosa 4, autosomal dominant) 337. GADD45Agrowth arrest and DNA-damage-inducible, alpha 338. LCATlecithin-cholesterol acyltransferase 339. GSR glutathione reductase 340.TOP2A topoisomerase (DNA) II alpha (170 kD) 341. GPX1 glutathioneperoxidase 1 342. FLT3 fms-related tyrosine kinase 3 343. CEBPBCCAAT/enhancer binding protein (C/EBP), beta 344. TPM1 tropomyosin 1(alpha) 345. ABCA4 ATP-binding cassette, sub-family A (ABC1), member 4346. KCNH2 potassium voltage-gated channel, subfamily H (eag- related),member 2 347. HNF4A hepatocyte nuclear factor 4, alpha 348. DPYDdihydropyrimidine dehydrogenase 349. MADH2 MAD, mothers againstdecapentaplegic homolog 2 (Drosophila) 350. AFP alpha-fetoprotein 351.TIMP2 tissue inhibitor of metalloproteinase 2 352. ITK IL2-inducibleT-cell kinase 353. ABL2 v-abl Abelson murine leukemia viral oncogenehomolog 2 (arg, Abelson-related gene) 354. SCYA4 small induciblecytokine A4 355. GCGR glucagon receptor 356. TCF3 transcription factor 3(E2A immunoglobulin enhancer binding factors E12/E47) 357. MYB v-mybmyeloblastosis viral oncogene homolog (avian) 358. LTA lymphotoxin alpha(TNF superfamily, member 1) 359. LIF leukemia inhibitory factor(cholinergic differentiation factor) 360. CYBB cytochrome b-245, betapolypeptide (chronic granulomatous disease) 361. CTSL cathepsin L 362.BCL2A1 BCL2-related protein A1 363. TFRC transferrin receptor (p90,CD71) 364. RALGDS ral guanine nucleotide dissociation stimulator 365.CYP2C8 cytochrome P450, subfamily IIC (mephenytoin 4- hydroxylase),polypeptide 8 366. CD38 CD38 antigen (p45) 367. PRKCZ protein kinase C,zeta 368. LAMR1 laminin receptor 1 (67 kD, ribosomal protein SA) 369.IL12A interleukin 12A (natural killer cell stimulatory factor 1,cytotoxic lymphocyte maturation factor 1, p35) 370. FGA fibrinogen, Aalpha polypeptide 371. EEF1A1 eukaryotic translation elongation factor 1alpha 1 372. CYP21A2 cytochrome P450, subfamily XXIA (steroid21-hydroxylase, congenital adrenal hyperplasia), polypeptide 2 373. CSF2colony stimulating factor 2 (granulocyte-macrophage) 374. TNFRSF5 tumornecrosis factor receptor superfamily, member 5 375. MBP myelin basicprotein 376. PTK2 PTK2 protein tyrosine kinase 2 377. KLK3 kallikrein 3,(prostate specific antigen) 378. GALT galactose-1-phosphateuridylyltransferase 379. APEX APEX nuclease (multifunctional DNA repairenzyme) 380. EPHB2 EphB2 381. BIK BCL2-interacting killer(apoptosis-inducing) 382. SLC2A1 solute carrier family 2 (facilitatedglucose transporter), member 1 383. IL2RA interleukin 2 receptor, alpha384. IFNGR2 interferon gamma receptor 2 (interferon gamma transducer 1)385. AXL AXL receptor tyrosine kinase 386. ADRB1 adrenergic, beta-1-,receptor 387. RAD51 RAD51 homolog (RecA homolog, E. coli) (S.cerevisiae) 388. GJA1 gap junction protein, alpha 1, 43 kD (connexin 43)389. EWSR1 Ewing sarcoma breakpoint region 1 390. CCR2 chemokine (C—Cmotif) receptor 2 391. RELA v-rel reticuloendotheliosis viral oncogenehomolog A, nuclear factor of kappa light polypeptide gene enhancer inB-cells 3, p65 (avian) 392. CTNNA1 catenin (cadherin-associatedprotein), alpha 1 (102 kD) 393. MYO7A myosin VIIA (Usher syndrome 1B(autosomal recessive, severe)) 394. F3 coagulation factor III(thromboplastin, tissue factor) 395. EPHX1 epoxide hydrolase 1,microsomal (xenobiotic) 396. CRK v-crk sarcoma virus CT10 oncogenehomolog (avian) 397. ENO1 enolase 1, (alpha) 398. TGFBR1 transforminggrowth factor, beta receptor I (activin A receptor type II-like kinase,53 kD) 399. RAC1 ras-related C3 botulinum toxin substrate 1 (rho family,small GTP binding protein Rac1) 400. ANPEP alanyl (membrane)aminopeptidase (aminopeptidase N, aminopeptidase M, microsomalaminopeptidase, CD13, p150)

A kit and/or method according to the invention may be used to detectinfectious diseases. Some infectious diseases are life threatening andcan appear in combination with other infections. Thus, the sooner theycan be detected and characterised, the sooner an appropriate therapy canbe established and better for the patient. A kit and/or method asdisclosed above can be used to detect said infectious agents. Componentswhich may be detected according to the kit and/or method are those whichform part of the infectious agent and/or are produced by the infectiousagent. Viruses in diseased individuals detectable according to the kitand/or method include, but are not limited to HCV, HIV, HBV, HTLV, HPV(see also oncology). Bacteria in diseased individuals detectableaccording to the kit and/or method include, but are not limited tomycobacteria, syphilis, Staphylococcus aureus (screening of MRSA).

A kit and/or method according to the invention may be used to detectneurodegenerative diseases. Components which may be detected are thoseinvolved in degenerative diseases and include, but are not limited tobeta-amyloids (Alzheimer's disease), hTAU, phosphoTAU and APOE.

A kit and/or method according to the invention may be used to detectprion-related diseases. Diseases associated with prions includeKreutzfeld Jacob disease and BSE.

A kit and/or method according to the invention may be used to detectdiseases related to autoimmunity. Components which may be detected arethose involved in autoimmunity include, but are not limited to ANA,Jo-1, Myeloperoxidase, RNP, Scl-70, Sm, SS-A.

A kit and/or method according to the invention may be used to detectdiseases related to allergy. Components which may be detected are thoseinvolved in allergy and include, but are not limited to IgE,IgG-subclasses and circulating antibodies.

A kit and/or method according to the invention may be used in the fieldof genomics to detect susceptibility to disease, possibility of passingconditions to offspring, single nucleotide polymorphisms etc. Examplesof fields in which a kit and/or method of the invention apply include,but are not limited to HLA typing, p53 polymorphism (SNP) related to thesensitivity of developing a cervix carcinoma after an HPV 16 infection,hypertension, detection of polymorphism in relation to thesusceptibility for osteoporosis, detection of mutations in Factor V(Leiden), detection of the genetic susceptibility for SIDS (cot death),hereditary: paternity tests, etc., detection of micro satelliteinstability, detection of the success rate of therapy related tocessation of smoking, detection of disturbances in the metabolism oflipids including cholesterol (HDL, LDL, VLDL and their receptors) inrelationship to cardiovascular diseases such as atheromathosis,detection of genomic defects related to obesitas, detection of genomicdefects related to diabetes, detection of mutations associated with drugresistance (to HIV, etc.), screening and detection of systic fibrosis(CFTR mutations), detection of mutations in the mitochondrial genomerelated to a number of diseases as: neurogenic muscular weakness,retinitis pigmentosa, ocular myopathy, etc.

A kit and/or method according to the invention may be used in the fieldsrelated to environmental testing. Many applications are related to waterwhere it is important to have a technology which is sensitive enough todetect very small amounts of contaminants or unwanted compounds inreasonably economical manner. Examples of environmental tests include:

checking (monitoring) of yeast infections in swimming pool water

monitoring of biological pollution in general

biological contaminants in potable water (amoebae, coliform bacteria,etc.).

In addition to water testing, the environmental testing for geneticallymodified organisms may be performed according to a kit and/or method ofthe present invention. Genetically Modified Organisms can be detected,or samples screened for the absence of. Checking for possiblemodifications is sometimes difficult, however, a sensitive techniquesuch as that provided by the present method is suitable for such apurpose.

A kit and/or method according to the invention may be used to detect theinfection of food. Inspection of all places and objects related to foodneeds sensitive methods, and kit and/or method of the present inventionprovide the required sensitivity. Furthermore, a kit and/or method ofthe present invention can be performed and the results obtained at thesite at which the inspection takes place, so obviating the need to sendsamples to a lab. Thus, steps can be taken immediately if necessary.Examples of the agents that may be detected include Listeria,Salmonella, prions (for BSE). Molecules which may be detected assay arethose which form part of the agent and/or are produced by the agent.

A kit and/or method of the present invention may be applied in standardbiochemical detection protocols. All the existing types of blottingtechniques show an enhancement in sensitivity using the method disclosedherein, without the requirement for radioisotopes or chemilluminescentdetection such as photographic plates, or phosphor screens. It is alsopossible to use the method in combination with image analysis. Examplesof blotting protocols that may use methods of the present inventioninclude but are not limited to Western blotting, Northern blotting,Southern blotting, vacuum blotting, contact blotting, reversed line blotand related techniques, dot blotting, micro-arrays, macro-arrays.

Another embodiment of the present invention is a method for stainingsections of cells and/or tissues suitable for visualisation usingmicroscopy. Types of microscopy may be any, and include, but are notlimited to light microscopy, tunneling electron microscopy, scanningelectron microscopy, transmission electron microscopy.

According to one aspect of the invention, a method for stainingcomponents in cell and/or tissue sections, said staining suitable forvisualisation using microscopy comprises the following steps:

n) incubating section with one or more tagged probes directed against acomponent,

m) incubating section with metal-particle-labeled anti-tag monoclonal orpolyclonal antibody, said antibody raised in species A,

o) incubating section with an antibody conjugate, said conjugatecomprising:

-   -   one or more antibodies, anti-A, directed against immunoglobulins        of species A,    -   one or more antibodies, anti-B, directed against immunoglobulins        of species B,        optionally one or more substances which directly or indirectly        cause a quantitative colour change,        p) incubating the section with a polypeptide capable recognition        by anti-B antibodies, said polypeptide labelled with one or more        substances which directly or indirectly cause a quantitative        colour change, and        q) optionally incubating the section in a metal enhancement        reagent and/or a colour change reagent that is a suitable        substrate of an enzyme attached to the antibody conjugate.

The antibody conjugate is already defined herein. The introduction ofother steps into the method, such as wash steps, for example, may beknown by the skilled artisan practicing in the field ofimmunohistochemistry. The “metal enhancement reagent” of step q) isalready defined herein for step i). The sections described above arevisualised by observing the dye, by observing the metal, by metalenhancement and/or by making use of the enzyme that catalyses the colourchange attached to the antibody conjugate. It is an aspect of theinvention to visualise the samples using metal enhancement alone. It isan aspect of the invention to visualise the samples by observing the dyealone. It is an aspect of the invention to visualise the samples byobserving the metal without metal enhancement. It is an aspect of theinvention to visualise the samples using only enzyme-linked colourchange. It is an aspect of the invention to visualise the samples usingone or more of the aforementioned visualisations.

In another aspect of the invention, the use of tagged probe as describedin a method above is circumvented by using an antibody raised in speciesA directly against a component in the section. Thus, in this embodimentof the invention, step n) as described above is absent, and step m)reads:

m) incubating section with metal-particle labeled anti-componentmonoclonal or polyclonal antibodies, said antibodies raised in speciesA.

Another embodiment of the present invention is a method for detecting acomponent in a section, said method as describe above, furthercomprising, after step p) the steps of:

p-1) repeating steps o) to p), and

p-2) optionally repeating step p-1).

The steps described in the embodiment above are an amplification, whichfurther enhances the sensitivity of the above method, while minimizingthe increase in background signal.

Another embodiment of the present invention is a kit for stainingsections of cells and/or tissues suitable for visualisation usingmicroscopy comprising the following components:

q) a container in which a quantity of antibody conjugate as describedabove is present.

A container may be any sealed or resealable vessel as describedelsewhere herein.

A kit for staining sections according to the present invention allows askilled artisan to perform one or more steps of the method disclosedherein, in a convenient manner. The kit may allow a method of thepresent invention to be performed without the need to measure ordetermine the concentrations of reagents, so enabling a fast andreproducible staining of sections.

Another aspect of the present invention is a kit for staining sectionsas disclosed herein, further comprising one or moremetal-particle-labelled probes. Each probe may be specific for acomponent in a section to be detected.

Another aspect of the present invention is a kit as disclosed herein,comprising antibody conjugate.

Another aspect of the present invention is a kit as disclosed herein,anti-tag antibody raised in species A, said antibody optionally labeledwith metal particle.

Another aspect of the present invention is a kit as disclosed hereincomprising one or more additional containers in which reagent necessaryfor visualisation using the colour change enzyme linked to the antibodyconjugate are present.

Another aspect of the present invention is a kit as disclosed hereincomprising one or more additional containers in which reagent necessaryfor performing metal enhancement are present.

Another aspect of the present invention is a kit as disclosed herein, apolypeptide capable recognition by anti-B antibodies, said polypeptidelabelled with one or more substances which directly or indirectly causea quantitative colour change.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which metal-particle-labelledanti-component antibodies are present.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which a tagged probe(s) capable ofbinding to a component(s) is present.

Another aspect of the invention is a kit as disclosed herein comprisingone or more additional containers in which a polypeptide capablerecognition by anti-B antibodies, said polypeptide labelled with one ormore substances which directly or indirectly cause a quantitative colourchange is present

In another aspect of the invention, the kit for staining sectionsenables the skilled person to perform one or more of the methoddisclosed herein. The kit may comprise one or more additional containersin which reagents are present enabling the skilled person to perform thecomplete method. Alternatively, the kit may comprise a minimum number ofcontainers, such as only item q), for example, that enables a skilledperson to perform the method disclosed herein.

In another aspect of the invention, the kit contains instructions foruse. In another aspect of the invention the instructions describe amethod of the invention as disclosed herein.

A method and/or kit of the present invention for staining sections ofcells may equally well be performed on any cell or tissue in theapplications of flow cytometry and in situ hybridisation, wherein thevisualisation of cells and tissues is necessary. Due to the sensitivityof the method as disclosed herein, target antigens (proteins and othersubstances) can be visualised in tissues and cells with antibodies and,using in situ hybridization, they can be visualised by use of nucleicacid probes.

EXAMPLES

Section 1: Materials and Methods

Oligonucleotides

Target: 5′ GGATTATTGTTAAATATTGATAAGGAT 3′ Visualisation5′ ATCCTTATCAATATT 3′ oligo: Oligo op drager: 5′ TAACAATAATCC 3′The above mentioned oligonucleotides are derived from the Anthrax lethalfactor genome.Nylon or Nitrocellulose Coated Slides

Coated slides such as Nytran coated slides, nitrocellulose coated slideswere purchased from Schleicher and Schuell and printed with DNAcapturing oligonucleotides using a Microcast micro-arrayer.

Mofified DNA oligonucleotides were custom made by Qbiogene (France) andin varying concentrations dissolved in printing buffer (6×SSC).

Micro-arrays were manufactured by printing varying oligonucleotideconcentrations ranging from 0.001 μM to 20 μM on the coated glass slide.Negative controls consisted of printing buffer without DNAoligonucleotides and printing buffer with a DNA oligonucleotidecomplementary to a non-related sequence in the same concentration as thecapturing oligonucleotide of interest. After drying during 30 minutes atroom temperature, the slides were baked at 80° C. during 30 minutes. DNAprinted at Nytran coated slides was cross linked using UV radiationaccording to previously described protocols.

Slides were stored dustfree at 4° C. until further analysis.

Silane Coated Slides

Coated slides such as Nytran coated slides, nitrocellulose coated slideswere purchased from Schleicher and Schuell and printed with DNAoligonucleotides using a Microcast micro-arrayer.

DNA oligonucleotides were custom made by Qbiogene (France) and invarying concentrations dissolved in printing buffer. Silane coatedslides and modified oligonucleotides were activated according topreviously described protocols.

Micro-arrays were manufactured by printing varying oligonucleotideconcentrations ranging from 0.001 μM to 20 μM on the activated silanecoated glass slide. After drying during 30 minutes at room temperature,the printed slides were stored dustfree at 4° C.

Classical Glass Slides

Classical microscopical glass slides were washed with bidistilled waterfollowed by immersion in a 10% NaOH solution at room temperature afteran ultrasound treatment during 30 minutes. After several washes inrunning tap water, slides were washed several times with bidistilledwater. Afterwards the slides were dried at 80° C.

Modified oligonucleotides were activated and coupled with aminosilaneaccording to previously described protocols (Kumar et al. Silanizednucleic acids: a general platform for DNA immobilization. Nucleic acidsres 2000; 28: e71.).

The silanized oligonucleotides were dissolved in printing buffer (50%DMSO,) and printed as described above.

Printing of the Micro-Arrays

Micro-arrays were printed with each concentration in sixfold, capturingoligonucleotides as well as the above described appropriate negativecontrols.

Section 2: Experiments to Compare Methods of Detection Using Gold andEnzyme Visualisations in Combination with Polymer Amplification andOther Technologies

Part 1—Visualization Experiments of Labeled Oligonucleotides Attached atthe Above Described Solid Assays

Experiment 1—part 1: Visualization of Labeled Oligonucleotides usingStreptavidin

Micro-arrays were printed as described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA (SERVA,Germany).

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/alkaline phosphatase(concentration 1/1000 in PBS/BSA buffer) (Roche Germany) during 60minutes followed by three washes with PBS/BSA buffer at roomtemperature.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer (Dako, Denmark) during 30minutes at room temperature.

Experiment 2—Part 1: Visualization of Labeled Oligonucleotides usingStreptavin Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution for 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA for 5 minutes.

Slides were incubated with streptavidin/gold 0.8 nm (concentration 1/50in washing buffer) or 6 nm (concentration 1/20 in washing buffer)(Aurion, the Netherlands) for 120 minutes followed by six washes withwashing buffer at room temperature.

Slides were rinsed triplefold for five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement (Aurion, theNetherlands) for 15 minutes.

Experiment 3—Part 1: Visualization of Labeled Oligonucleotides usingMonoclonal Antibodies Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution for 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) for 5 minutes.

Slides were incubated with monoclonal antibody/gold 0.8 nm(concentration 1/50 in washing buffer), 6 nm (concentration 1/20 inwashing buffer) or 40 nm during 120 minutes followed by six washes withwashing buffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 4—Part 1: Visualization of Labeled Oligonucleotides usingPolyclonal Antibodies Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal antibody/gold 0.8 nm(concentration 1/50 in washing buffer) or 6 nm (concentration 1/20 inwashing buffer) during 120 minutes followed by six washes with washingbuffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 5—Part 1: Visualization of Labeled Oligonucleotides usingMonoclonal and Polyclonal Antibodies in a Polymer Enhanced AmplificationTechnique Visualized Enzymatically

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies and labeled withalkaline phosphatase enzym for 30 minutes followed by six washes withwashing buffer.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Experiment 6—Part 1: Visualization of Labeled Oligonucleotides usingGold Labeled Monoclonal and Polyclonal Antibodies in a Polymer EnhancedAmplification Technique

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies for 30 minutes followedby six washes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 7—Part 1: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized Enzymatically

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were incubated with streptavidin/alkaline phosphatase(concentration 1/1000 in PBS/BSA buffer) during 60 minutes followed bythree washes with PBS/BSA buffer at room temperature.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Experiment 8—Part 1: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized with Gold Labeled Streptavidin

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were incubated with streptavidin/gold 0.8 nm (concentration 1/50in washing buffer) or 6 nm (concentration 1/20 in washing buffer) during120 minutes followed by six washes with washing buffer at roomtemperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 9—Part 1: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized with Gold Labeled Monoclonal orPolyclonal Antibodies

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 120 minutesfollowed by six washes with washingbuffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Part 2: Hybridization Experiments of Labeled Oligonucleotides Attachedat the Above Described Solid Assays

Experiment 2.1—Part 2: Visualization of Labeled Oligonucleotides usingStreptavidin

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold includingadequate positive and negative controls. Micro-arrays were baked at 80°C. during 30 minutes and stored dust free at 4° C. until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide in a concentration of 250 ng/ml hybridization mixture.Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/alkaline phosphatase(concentration 1/1000 in PBS/BSA buffer) during 60 minutes followed bythree washes with PBS/BSA buffer at room temperature.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Experiment 2.2—Part 2: Visualization of Labeled Oligonucleotides usingStreptavin Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with streptavidin/gold 0.8 nm (concentration 1/50in washing buffer) or 6 nm (concentration 1/20 in washing buffer) during120 minutes followed by six washes with washing buffer at roomtemperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 3—Part 2: Visualization of Labeled Oligonucleotides usingMonoclonal Antibodies Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dustfree at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal antibody/gold 0.8 nm(concentration 1/50 in washing buffer) or 6 nm (concentration 1/20 inwashing buffer), or 40 nm for 120 minutes followed by six washes withwashing buffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 4—Part 2: Visualization of Labeled Oligonucleotides usingPolyclonal Antibodies Labeled with Gold Particles

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal antibody/gold 0.8 nm(concentration 1/50 in washing buffer) or 6 nm (concentration 1/20 inwashing buffer) during 120 minutes followed by six washes with washingbuffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 5—Part 2: Visualization of Labeled Oligonucleotides usingMonoclonal and Polyclonal Antibodies in a Polymer Enhanced AmplificationTechnique Visualized Enzymatically

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies and labeled withalkaline phosphatase enzyme for 30 minutes followed by six washes withwashing buffer.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Experiment 6—Part 2: Visualization of Labeled Oligonucleotides usingGold Labeled Monoclonal and Polyclonal Antibodies in a Polymer EnhancedAmplification Technique

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies for 30 minutes followedby six washes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 7—Part 2: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized Enzymatically

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were incubated with streptavidin/alkaline phosphatase(concentration 1/1000 in PBS/BSA buffer) during 60 minutes followed bythree washes with PBS/BSA buffer at room temperature.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Experiment 8—Part 2: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized with Gold Labeled Streptavidin

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were incubated with streptavidin/gold 0.8 nm (concentration 1/50in washing buffer) or 6 nm (concentration 1/20 in washing buffer) during120 minutes followed by six washes with washing buffer at roomtemperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Experiment 9—Part 2: Visualization of Labeled Oligonucleotides usingCARD Amplification Technology Visualized with Gold Labeled Monoclonal orPolyclonal Antibodies

Micro-arrays were printed like described previously. All concentrationsranging from 0.001 μM to 20 μM were spotted in sixfold. Micro-arrayswere baked at 80° C. during 30 minutes and stored dust-free at 4° C.until use.

The slides were prehybridized with hybridization mixture supplementedwith sonicated herring sperm DNA (150 μg/5 ml hybridization mixture) for2 hours at room temperature.

Hybridization assay was set up using target DNA and/or visualisationoligonucleotide consisting of labeled oligonucleotide in a concentrationof 250 ng/ml hybridization mixture.

Hybridization was carried out overnight at 37° C.

Slides were washed twice with 2×SSC at room temperature

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with wash buffer during 5 minutes.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 120 minutesfollowed by six washes with washing buffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Results and Discussion

Results of Experiments without Signal Amplification:

Experiment 1—Visualization with Streptavidin-Enzyme

Hybridization was visualized as deep red or dark brown spots accordingto the used enzyme and substrate. The spots were easy discernible at thewhite background of the slide.

Background staining was not present. Visualization was achieved withspotted molecular probe concentration ranging from 0.2 mM to 0.02 mM.

Experiment 2—Visualization with Streptavidin Gold 0.8 nm and 6 nm

Hybridization was visualized as deep black grey spots according to theused silver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was not presented.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.02 mM.

Experiment 3—Visualization with Monoclonal Mouse Antibodies with Gold0.8 nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was not present.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method. In comparison with the polyclonal antibodiesthe signal was slightly sharper.

Experiment 3—Visualization with Monoclonal Mouse Antibodies with Gold 40nm

Hybridization was faintly visualized as light rosa spots only at thehighest spot concentration of 0.2 mM. Silver enhancement did not yieldsignificant signal strenghtening. Background staining was considerable.

Experiment 4—Visualization with Polyclonal Goat Antibodies with Gold 0.8nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was not present.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method.

Results of Experiments with Signal Amplification—CARD-Amplification withShort Spacer:

Experiment 7—Visualization with Streptavidin-Enzyme

Hybridization was visualized as deep red or dark brown spots accordingto the used enzym and substrate. The spots were easy discernible at thewhite background of the slide. Background staining was moderate tostrong. Visualization was achieved with spotted molecular probeconcentration ranging from 0.2-mM to 0.0002 mM.

Experiment 8—Visualization with Streptavidin Gold 0.8 nm and 6 nm

Hybridization was visualized as deep black grey spots according to theused silver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was moderate. Visualizationwas achieved with spotted molecular probe concentration ranging from 0.2mM to 0.0002 mM.

Experiment 9—Visualization with Polyclonal Goat Antibodies with Gold 0.8nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement strategy. The spots were easy discernible at thewhite background of the slide. Background staining was moderate.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.0002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method.

Experiment 9—Visualization with Monoclonal Mouse Antibodies with Gold0.8 nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was moderately present.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method. In comparison with the polyclonal antibodiesthe signal was slightly sharper.

Experiment 9—Visualization with Monoclonal Mouse Antibodies with Gold 40nm

Hybridization was faintly visualized as light brown spots only at thehighest spot concentration of 0.2 mM. Silver enhancement did not yieldsignificant signal enhancement. Background staining was considerable.

Results of Experiments with Signal Amplification—CARD-Amplification withLong Spacer:

Experiment 7—Visualization with Streptavidin-Enzyme

Hybridization was visualized as deep red or dark brown spots accordingto the used enzyme and substrate. The spots were easy discernible at thewhite background of the slide. Background staining was moderate.Visualization was acieved with spotted molecular probe concentrationranging from 0.2 mM to 0.0002 mM.

Experiment 8—Visualization with Streptavidin Gold 0.8 nm and 6 nm

Hybridization was visualized as deep black grey spots according to theused silver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was slight. Visualizationwas achieved with spotted molecular probe concentration ranging from 0.2mM to 0.0002 mM.

Experiment 9—Visualization with Polyclonal Goat Antibodies with Gold 0.8nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was very slight.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.0002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method.

Experiment 9—Visualization with Monoclonal Mouse Antibodies with Gold0.8 nm and 6 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was very slight.Visualization was achieved with spotted molecular probe concentrationranging from 0.2 mM to 0.002 mM. However the signal was stronger withthe 6 nm gold compared to the 0.8 nm gold and sharper than with thestreptavidin-gold method. In comparison with the polyclonal antibodiesthe signal was slightly sharper.

Experiment 9—Visualization with Monoclonal Mouse Antibodies with Gold 40nm

Hybridization was faintly visualized as light brown spots only at thehighest spot concentration of 0.2 mM. Silver enhancement did not yieldsignificant signal strenghtening. Background staining was considerable.

Results of Experiments with Signal Amplification—Amplification usingPolymertechnology:

Experiment 5—Visualization with Monoclonal Antibodies/Polymer Labeledwith Alkaline Phosphatase

Hybridization was visualized as deep red spots. The spots were easydiscernible at the white background of the slide. Background stainingwas slight. Visualization was achieved with spotted molecular probeconcentration ranging from 0.1 mM to 0.0001 mM.

Experiment 6—Visualization with Polyclonal Goat Antibodies with Gold 0.8nm, 6 nm and 40 nm/Polymer/Monoclonal Mouse Antibodies 0.8 nm, 6 nm and40 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was very slight.Visualization was achieved with spotted molecular probe concentrationranging from 0.1 mM to 0.0001 mM. However the signal was stronger withthe 0.8 nm gold compared to the 6 nm gold and sharper than with thestreptavidin-enzym method. The 40 nm gold particles gave a slight rosareaction barely discernible with the naked eye.

Experiment 6—Visualization with Monoclonal Mouse Antibodies 0.8 nm, 6nm, 40 nm/Polymer/Polyclonal Goat Antibodies with Gold 0.8 nm, 6 nm and40 nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was not present.Visualization was achieved with spotted molecular probe concentrationranging from 0.1 mM to 0.0001 mM. However the signal was stronger withthe 0.8 nm gold compared to the 6 nm gold and sharper than with thestreptavidin-enzyme method. The 40 nm gold particles gave a slight rosareaction barely discernible with the naked eye.

Experiment 6—Visualization with Monoclonal Goat Antibodies with Gold 0.8nm, 6 nm, 40 nm/Polymer/Monoclonal Mouse Antibodies 0.8 nm, 6 nm and 40nm

Hybridization was visualized as deep black spots according to the usedsilver enhancement. The spots were easy discernible at the whitebackground of the slide. Background staining was very slight.Visualization was achieved with spotted molecular probe concentrationranging from 0.1 mM to 0.0001 mM. However the signal was stronger withthe 0.8 nm gold compared to the 6 nm gold and sharper than with thestreptavidin-enzym method. The 40 nm gold particles gave a slight rosareaction barely discernible with the naked eye.

Experiment 6—Visualization with Monoclonal Mouse Antibodies with Gold 40nm/Polymer/Monoclonal Mouse Antibodies with Gold 40 nm

Hybridization was faintly visualized as light red spots only at thehighest spot concentration of 0.2 mM. Silver enhancement did not yieldsignificant signal strengthening. Background staining was considerable.

Section 3: Detection and Subtyping of HPV DNA

Micro-arrays were printed like described previously using specificoligonucleotides detecting HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV52 and HPV 58. The oligonucleotides were dissolved in printing bufferresulting in an end concentration of 10 μM and were spotted in sixfoldincluding adequate positive and negative controls using a Microcastmicro-arrayer.

This results in a micro-array with 7 seven rows, each row consisting ofsix identical spots, representing the seven above described HPV types.The negative controls consisted of printing buffer without DNA and asecond negative control consisted of an oligonucleotide coding for anon-related gene segment and were printed as two rows consisting of sixspots where one row consisted out of printing buffer without DNA andanother row consisted of non-related DNA oligonucleotide.

The positive control consisted of an equimolar mixture of the abovedescribed HPV type specific oligonucleotides printed in one rowconsisting of six identical spots.

After printing the micro-arrays were air dried at room temperature for15 minutes.

Micro-arrays were baked at 80° C. during 30 minutes and stored dust-freeat 4° C. until use.

Hybridization with PCR Labeled Amplification Product

Hybridization assay was set up using PCR amplified HPV DNA.

During the PCR reaction the amplification product was labeled using abiotin labeled primer.

In another experiment the PCR was set up with two unlabeled primers.

Ten microliter of the PCR amplification product was denatured with 10 μldenaturation solution (NAOH/EDTA). The denatured DNA solution was addedto 2 ml of hybridization mixture. The micro-arrays were covered with acover slip and hybridized overnight at 37° C. in a humid chamber.

Slides were washed with SSC buffer.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

The hybridization of the biotin labeled PCR product was revealed usingone of the methods described above.

Visualisation with Streptavidin-Alkaline Phosphatase

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/alkaline phosphatase(concentration 1/1000 in PBS/BSA buffer) during 60 minutes followed bythree washes with PBS/BSA buffer at room temperature.

The alkaline phosphatase reaction was developed by incubating the slideswith napthol substrate in appropriate buffer during 30 minutes at roomtemperature.

Visualisation with Streptavidin-Gold

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with streptavidin/gold 0.8 nm (concentration 1/50in washing buffer) or 6 nm (concentration 1/20 in washing buffer) during120 minutes followed by six washes with washing buffer at roomtemperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Visualisation with Mono-or Polyclonal Anti-Biotin Antibody

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal or polyclonal antibody/gold 0.8 nm(concentration 1/50 in washing buffer) or 6 nm (concentration 1/20 inwashing buffer) during 120 minutes followed by six washes with washingbuffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Visualisation using Signal Amplification with Polymer Technology

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies for 30 minutes followedby six washes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Visualisation using Signal Amplification with CARD Technology

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were incubated with streptavidin/peroxidases (concentration 1/500in PBS/BSA buffer) during 60 minutes followed by three washes withPBS/BSA buffer at room temperature.

The slide was incubated with biotinylated tyramine diluted 1/50 in PBSsolution supplemented with 0.03% H₂O₂ for 10 minutes followed by threewashes with washbuffer during 5 minutes.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 120 minutesfollowed by six washes with washing buffer at room temperature.

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes

Hybridization with Unlabeled PCR Amplification Product, Visualizationusing Anti-DNA Antibody, Signal Amplification with Polymer Technologyand Gold.

Hybridization assay was set up using PCR amplified HPV DNA.

In this part of the experiment the PCR was set up with two unlabeledprimers.

Ten microliter of the PCR amplification product was denatured with 10 μldenaturation solution (NAOH/EDTA). The denatured DNA solution was addedto 2 ml of hybridization mixture. The micro-arrays were covered with acover slip and hybridized overnight at 37° C. in a humid chamber.

Slides were washed twice with 2×SSC supplemented with 0.1% SDS at roomtemperature.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

The hybridization of the unlabeled PCR product with its captureoligonucleotide at the micro-array was revealed using an anti-DNAantibody.

Slides were washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with anti-DNA during 60 minutes followed by sixwashes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with anti-mouseantibodies and anti-rabbit antibodies for 30 minutes followed by sixwashes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labeledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

Results and Discussion

Hybridized micro-arrays showed areas with very sharp black or redcoloured spots in some areas depending on the used substrate. Otherareas did not show any signal. Background signal was completely absent.

Hybridization was revealed as easy discernible bright spots coloureddeep red when using the alkaline phophatase technique for visualizationor as deep grey to black spots when using gold-silver enhancementtechnology. The results were easily evaluated with the naked eye. Thebest results were obtained with gold labeled antibodies used in apolymer amplification technique.

Negative controls did not show any sign of positivity. The positivecontrol was strongly positive. In one experiment the presence of HPV 16was revealed and in another experiment the presence of HPV 18 washighlighted. Cross-hybridization with other molecular probes was notnoted. Samples without HPV DNA did not give a signal at the micro-array.

Section 4: Application of Immunohistochemistry: Visualization of TissueAntigens with of Monoclonal Antibodies using Gold Labeled Monoclonal andPolyclonal Antibodies in a Polymer Enhanced Amplification Technique

Sections of 5 μm were cut from paraffin embedded formalin fixed tissueof squamous lung carcinoma and adhered to poly-l-lysine coated glassslides, dried at 55° C. overnight and stored dust-free at roomtemperature. The sections were deparaffinized in two rinses of xylenesubstitute, followed by rehydratation in an descending series ofalcohols down to deionized water. Slides were washed twice with PBS (pH7.4) and incubated with anti-ema (epithelial membrane antigen)monoclonal mouse antibody (Dakopatts Denmark) according to theinstructions of the manufacturer). Slides were washed twice with PBS (pH7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with anti-mouseantibodies and anti-rabbit antibodies for 30 minutes followed by sixwashes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes.

The results showed clear and sharp staining with excellent contrast.

Section 5

5.1 Detection of Specific Antibodies

Micro-arrays were printed as described previously using specificantigens such as CEA (carcino-embronal antigen) and EMA (epithelialmembrane antigen). The antigens were diluted in printing bufferconsisting of PBS (pH 7.4) resulting in various concentrations and werespotted in sixfold including adequate positive and negative controlsusing a Microcast micro-arrayer.

This results in a micro-array with 7 seven rows, each row consisting ofsix identical spots, representing various concentrations of the abovedescribed antibodies or antigens. The negative controls consisted ofprinting buffer without antigen and a second negative control consistedof a non-related antigen and were printed as two rows consisting of sixspots where one row consisted out of printing buffer without antibody orantigen and another row consisted of non-related antigen.

The positive control consisted of an equimolar mixture ofantibodiesdirected against the above described antigens printed in onerow consisting of six identical spots.

After printing the micro-arrays were air dried at room temperature for15 minutes and stored dust-free at 4° C. until use.

Slides were washed twice with PBS (pH 7.4) supplemented with 3% BSAfollowed by an incubation during 30 minutes at room temperature followedby incubation with anti-CEA (carcino-embryonal antigen) antibody. Slideswere washed twice with PBS (pH 7.4) supplemented with BSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washingbuffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies for 30 minutes followedby six washes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement during 15minutes. The results showed clear and sharp staining with excellentcontrast.

5.2 Detection of Specific Antigens

Micro-arrays were printed like described previously using specificantibodies detecting CEA.

The antibodies were diluted in printing buffer consisting of PBS (pH7.4) resulting in various concentrations and were spotted in sixfoldincluding adequate positive and negative controls using a Microcastmicro-arrayer.

This results in a micro-array with 7 seven rows, each row consisting ofsix identical spots, representing various concentrations of the abovedescribed antibodies. The negative controls consisted of printing bufferwithout antibody or antigen and a second negative control consisted of anon-related antigen d were printed as two rows consisting of six spotswhere one row consisted out of printing buffer without antibody orantigen and another row consisted of non-related antibody or antigen.

The positive control consisted of an equimolar mixture of the abovedescribed antigen printed in one row consisting of six identical spots.

After printing the micro-arrays were air dried at room temperature for15 minutes and stored dust free at 4° C. until use.

Slides were washed twice with PBS (pH 7.4) and incubated with CEAantigen. Slides were washed twice with PBS (pH 7.4) supplemented withBSA.

Slides were incubated with the same PBS/BSA buffer solution during 30minutes at room temperature.

Slides were washed twice with special washing buffer (PBS pH 7.4supplemented with BSA) during 5 minutes and incubated with the samewashing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature.

Slides were incubated with dextran polymer coated with numerousanti-mouse antibodies and anti-rabbit antibodies for 30 minutes followedby six washes with washing buffer.

Slides were incubated with monoclonal or polyclonal antibody labelledwith gold particles ranging from 0.6 nm to 40 nm during 60 minutesfollowed by six washes with washing buffer at room temperature

Slides were rinsed triplefold during five minutes with PBS, followed bydistilled water.

The gold particles were visualized by silver enhancement for 15 minutes.

The results showed clear and sharp staining with excellent contrast.

1. A method for quantitatively and/or qualitatively detecting one ormore components in one or more samples, said component capable ofbinding to a probe, comprising the steps in the following order: a)applying onto a solid support i) one or more samples comprisingcomponents to be detected or ii) one or more probes, b) incubating thesolid support of step a) with: i) one or more tagged probes, in the casewhere the solid support has one or more samples comprising components tobe detected applied thereon, or ii) sample comprising tag-labeledcomponents to be detected, in the case where the solid support has oneor more probes applied thereon, said incubating followed by a wash step,c) incubating the solid support with a monoclonal or polyclonal antibodydirected against the tag of step b), said antibody raised in species Aand said antibody is labeled with metal particles of average diameterbetween 0.6 nm and 40 nm, followed by a wash step, d) incubating thesolid support with an antibody conjugate, said conjugate comprising: oneor more antibodies, anti-A, directed against immunoglobulins of speciesA, and one or more antibodies, anti-B, directed against immunoglobulinsof species B, said incubating followed by a wash step, e) incubating thesolid support with a polypeptide capable of recognition by anti-Bantibodies, said polypeptide labeled with one or more substances whichdirectly or indirectly cause a quantitative color change compared withthe solid support, f) incubating the solid support with a metalenhancement reagent, and g) reading the solid support to quantitativelyand/or qualitatively detect said components.
 2. The method according toclaim 1 wherein step a) comprises applying one or more probes onto thesolid support, and step b) comprises incubating the solid support withtag-labeled components.
 3. The method according to claim 1 wherein stepb) comprises incubating the solid support with the tag-labeledcomponents to be detected and wherein said antibody in step c) comprisesa metal-particle-labeled anti-component monoclonal or polyclonalantibody, said antibody raised in species A.
 4. The method according toclaim 1 further comprising the steps, after step e), of: e-1) repeatingsteps d) to e), and e-2) optionally repeating step e-1).
 5. The methodaccording to claim 2 wherein the solid support is supplied with theprobe pre-applied.
 6. The method according to claim 1 wherein thereading of step g) comprises the use of a color chart.
 7. The methodaccording to claim 1 wherein the reading of step g) comprises the use ofa device suitable for detecting changes in conductance and/or currentacross the solid support at positions at which said samples are applied.8. The method according to claim 1 wherein said tag is biotin.
 9. Themethod according to claim 1 wherein said polypeptide capable ofrecognition by anti-B antibodies is labeled with gold particles and/oralkaline phosphatase.
 10. The method according to claim 1, furthercomprising storing the solid support of step a) at a temperature between0 and 10 degrees Celsius.
 11. The method according to claim 1, whereinthe antibody conjugate further comprises one or more substances whichdirectly or indirectly cause a quantitative color change compared withthe solid support.
 12. The method according to claim 1, furthercomprising incubating the solid support with a color change reagent thatis a suitable substrate of an enzyme attached to the antibody conjugateafter step f).
 13. The method according to claim 1, wherein the metalparticle is gold.